Protein-DNA recognition is central to many biological processes. This interaction can be classified into two major types: sequence-specific and structure-specific. DNA-repair proteins generally do not recognize specific DNA sequences but search for unusual DNA structures which require repair. In addition, the DNA repair process often requires the concerted actions of several proteins. Our understanding of the structural biology of DNA-repair process is still in its infancy. A number of major DNA repair pathways have not been characterized at the molecular level. The mechanism of structure-specific DNA recognition as well as the interactions between proteins in the repair complex is not well understood. In this proposal, we plan to characterize the major pathway for double-strand DNA breaks repair. Double-strand DNA breaks are caused by ionizing radiation, V (D) J recombination and physiological oxidation reactions, and proper repair is important to maintain genomic stability. The proteins that are central to the major pathway of double-strand-break repair process are Ku70 (69.8 kD) and Ku80 (82.7 kD). These two proteins form a tight complex, bind to broken DNA ends and recruit other proteins, including a DNA-dependent protein kinase and a DNA-ligase to form the DNA repair machinery. Previous studies suggest that the Ku proteins contain structurally and functionally independent domains. We will analyze the structures of Ku domains and study the mechanism of the interactions between these domains, and their interactions with DNA and other proteins in the DNA-repair machinery using a combination of nuclear magnetic resonance (NMR) and molecular biological approaches. These studies will provide an understanding, at the molecular level, of the assembly of the proteins at DNA double strand break sites. This information is necessary to improve our knowledge of the molecular mechanism of the Ku-mediated DNA double-strand break repair pathway. This study should also improve our understanding of the structural biology of DNA repair in general.